The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor belongs to the glucagon/secretin/vasoactive intestinal polypeptide (VIP) receptor family. We mutated and deleted an amino acid residue (E261) which is located within the second intracellular loop of the rat PACAP type I receptor and which is highly conserved among the receptor family. The wild-type receptor and the mutant receptors were efficiently expressed at the surface of COS-7 cells at nearly the same level and revealed the same high affinity for the agonist PACAP-27. The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor were 4.6-, 5.7-, and 6.7-fold higher as compared with COS cells transfected with the wild-type receptor. Thus, all the mutant PACAP receptors were constitutively active. The data suggest that the glutamic acid in the second intracellular loop of the PACAP receptor may be a key residue to constrain the receptor in the inactive conformation with respect to its coupling to Gs proteins.
